Due to the recent rise in multi-drug resistant organisms, we investigated the potential for developing existing validated natural product scaffolds to produce new semi synthetic antibiotics with more potent activity. We focused on the low molecular weight aminocyclitol spectinomycin as it: has been under studied; has a safe pharmacological profile; targets a ribosomal binding site highly conserved across bacterial pathogens, but separate from other aminoglycosides; is potent in cell free assays; is structurally tractable, allowing for advanced synthetic modification using structure based drug design techniques; has limited clinical use due to bacterial penetration and uptake issues. Several novel analogs were generated and screened against panels of both sensitive and drug resistant bacteria to find compounds with improved anti-bacterial activity. From these series, we discovered a novel class of anti-tuberculosis agents, the spectinamides. The most potent of our compounds demonstrate: excellent anti-tuberculosis activity; on target inhibition of protein synthesis in M. tuberculosis; no cross resistance to existing anti-tuberculosis drugs including those active at the ribosome (Streptomycin, Kanamycin, Capreomycin and Linezolid); good pharmacokinetic profiles including excellent serum and microsomal stability; and anti- tuberculosis activity in vivo. Preliminary results show that improved activity of spectinamides results in part from superior uptake into TB bacilli. We believe the spectinamides are an important discovery of a new chemotype that can be used for the treatment of drug resistant M. tuberculosis infections which we plan to expand and further develop through this collaborative research proposal in three research aims: (i) Compound development - computer aided drug design will be used in an iterative drug development cycle for the design and synthesis of spectinamides with high anti-tubercular potency; (ii) Mode of action - Molecular techniques, including whole genome sequencing, will be used to determine the genetic basis for the improved activity and uptake of the spectinamides against TB; biochemical assays will be used evaluate mode of action of lead compounds at the target level to ensure compounds that progress in our lead development cycle remain potent and selective for inhibition of bacterial protein synthesis; (iii) Lead development and characterization - Compounds synthesized will progress through three stages of tests that include microbial assessment, pharmacokinetic testing, toxicologic and in vivo efficacy experiments. After each stage, the data will be used in further design and synthesis cycles in aim 1 and the best compounds will be selected to move on to the next stage such that viable, well characterized drug candidates will emerge from this study. In this study we propose to develop a novel class of protein synthesis inhibitors to treat problematic multi-drug resistant tuberculosis infections. Compounds discovered in this study may also have the potential to treat other drug resistant bacterial diseases.